Biophysical studies of microtubules

Microtubules exhibit dynamic instability, switching between persistent states of elongation and shortening at their ends. The GTP cap model proposes that a terminal crown of GTP-tubulin stabilizes the microtubule lattice and promotes elongation, while loss of this GTP cap converts the microtubule end to shortening.; We tested this hypothesis by mechanically cutting off the GTP cap with a glass microneedle, while observing them under video-enhanced differential interference contrast (VE-DIC) light microscopy. Our results showed a significant percentage of unstable plus ends and stable minus ends after cutting. This implies the existence of a metastable intermediate state between the elongation and shortening states.; The GTP cap model also predicts that tubulin dissociation from shortening ends is a two-step process where the average lengths of curved GDP-tubulin protofilaments will depend on the ratio of the protofilament peeling rate versus the protofilament breakage rate.; We tested this model by systematically increasing microtubule shortening rates by isothermal dilution in buffer containing various concentration of Mg{dollar}sp{lcub}+2{rcub}{dollar} and Ca{dollar}sp{lcub}+2{rcub}{dollar} ions. At above 20 mM Mg{dollar}sp{lcub}+2{rcub}{dollar} or 5 mM Ca{dollar}sp{lcub}+2{rcub}{dollar}, "knobs" were visible at the depolymerizing ends viewed under VE-DIC microscopy. The "knobs" were revealed to be "blossoms" of curved GDP-tubulin protofilaments by negative stain electron microscopy. Monte-Carlo simulations showed that the ratio of the peeling to breakage rates can control the steady-state average length of curved protofilaments.; Finally, the flexural rigidity of microtubules was measured using a laser trap. Kinesin-coated beads were allowed to bind to the microtubule nucleated from axonemes. The laser trap was used to trap the beads and bend the microtubules. Then the force of the trap was decreased until the bent microtubule sprung from the trap back to its original position. The flexural rigidity of the microtubule was calculated from the trap force at microtubule release and the geometry of the bend microtubule recorded by VE-DIC microscopy. We measured stiffness values of 6.8 {dollar}cdot{dollar} 10{dollar}sp{lcub}-24{rcub}{dollar} Nm{dollar}sp2{dollar} for the core GDP-tubulin lattice of microtubules, 2.4 {dollar}cdot{dollar} 10{dollar}sp{lcub}-24{rcub}{dollar} Nm{dollar}sp2{dollar} for taxol-bounded GDP-tubulin microtubule lattice, 13 {dollar}cdot{dollar} 10{dollar}sp{lcub}-24{rcub}{dollar} Nm{dollar}sp2{dollar} for GMPCPP-tubulin microtubules, and 50 {dollar}cdot{dollar} 10{dollar}sp{lcub}-24{rcub}{dollar} Nm{dollar}sp2{dollar} for MAPs-bounded GDP-tubulin microtubules.